Toll Like Receptors on Conjunctival Epithelium

描述（由申请人提供）：我们的目标是研究toll样受体（TLR）在人结膜上皮上的表达，并确定微生物配体激活TLR对眼部过敏性炎症的潜在贡献。研究结膜上皮细胞作为免疫调节剂的研究主要集中在促炎细胞因子的作用上，而微生物产物的直接作用尚未得到研究。初步研究表明，结膜上皮细胞表达一种模式识别受体TLR2，并通过介质释放和表面受体上调对金黄色葡萄球菌肽聚糖（SA-PGN）做出反应。结膜上皮细胞可能经常通过tlr和肥大细胞介质同时激活，这可能有助于慢性眼部变应性疾病的发生。第一个具体目标将集中于确定结膜上皮细胞表达的TLR的库（重点关注TLR1,2,4,6,9和辅助受体CD14），以及表达的TLR是否可被细胞因子和/或TLR配体诱导（TLR1,2和6的SA-PGN和酵母酶；TLR4的铜绿假单胞菌LPS， TLR9的CpG DNA）。这将包括通过印象细胞学对从人类受试者眼表获得的细胞（正常与各种疾病状态）进行体内检查。第二个特定目标将研究TLR配体激活结膜上皮细胞，微生物产物与各自TLR和信号（辅助蛋白MyD88， MD2和TIRAP）的特异性结合。激活将被定义为ICAM-1和/或HLA-DR表达上调和/或TNFalpha、IL-6、IL-8和/或RANTES的释放增强。第三个具体目标是研究TLR配体和ige活化纯化结膜肥大细胞的上清同时和顺序激活结膜上皮细胞的影响。联合激活的结果将与单独使用TLR配体或ige激活的肥大细胞上细胞的激活进行比较。结膜上皮细胞和肥大细胞将从尸体结膜组织中获得。评估蛋白表达的技术将包括流式细胞术、Western blot、免疫pcr（印迹细胞学细胞）和ELISA。评估mRNA表达的技术将包括Northern blot和RT-PCR（印迹细胞学细胞）。检测结合特异性和信号复合物的技术将包括阻断抗体、共免疫沉淀和凝胶移位测定。

DESCRIPTION (provided by applicant): Our goal is to investigate expression of Toll-like receptors (TLR) on human conjunctival epithelium and determine the potential contributions of TLR activation by microbial ligands to ocular allergic inflammation. Studies examining conjunctival epithelial cells as immunomodulators have been focused on the effects of pro-inflammatory cytokines, while the direct effects of microbial products have not been examined. Preliminary studies suggest that conjunctival epithelial cells express a pattern recognition receptor, TLR2, and respond to Staphylococcus aureus peptidoglycan (SA-PGN) with mediator release and surface receptor upregulation. It is likely that simultaneous activation of conjunctival epithelial cells via TLRs and mast cell mediators is a frequent occurrence, which may contribute to the development of chronic ocular allergic disease. The first specific aim will focus on determining the repertoire of TLRs (focusing on TLR1,2,4,6,9 and accessory receptor, CD14) expressed by conjunctival epithelial cells and whether the TLRs expressed are inducible by cytokines and/or TLR ligands (SA-PGN and yeast zymosan for TLRs 1,2, and 6; Pseudomonas aeruginosa LPS for TLR4, CpG DNA for TLR9). This will include in vivo examination of cells obtained from the ocular surface of human subjects (normal vs various disease states) via impression cytology. The second specific aim will investigate activation of conjunctival epithelial cells with TLR ligands, specific binding of microbial products to their respective TLRs and signaling (accessory proteins MyD88, MD2, and TIRAP). Activation will be defined as upregulation of ICAM-1 and/or HLA-DR expression and/or enhanced release of TNFalpha, IL-6, IL-8, and/or RANTES. The third specific aim will examine the effect of simultaneous and sequential activation of conjunctival epithelial cells with TLR ligands and supernates from IgE-activated purified conjunctival mast cells. The results of combined activation will be compared to activation with either TLR ligands or IgE-activated mast cell supernates alone. Conjunctival epithelial and mast cells will be obtained from cadaveric conjunctival tissues. Techniques to evaluate protein expression will include flow cytometry, Western blot, Immuno-PCR (in impression cytology cells), and ELISA. Techniques to evaluate mRNA expression will include Northern blot and RT-PCR (in impression cytology cells). Techniques to examine binding specificity and signaling complexes will include blocking antibodies, co-immunoprecipitation and gel shift assays.